Protein-digesting enzymes of papaya and pineapple

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* JAN 7 1942 *

Circular No. 631

December 1941 • Washington,

UNITED STATES DEPARTMENT OF AGRICULTURE

Protein-Digesting Enzymes of Papaya and

Pineapple

By A. K. Balls, head chemist, Enzyme Research Laboratory, Bureau of
Agricultural Chemistry and Engineering.

CONTENTS

Introduction 1

Sources of papain and bromelin 2

Preparation of papain 3

Crystalline papain 5

Page

Properties and uses of papain 6

Tenderizing meat 7

Bromelin 8

Summary 9

INTRODUCTION

Within animals and plants the chemical reactions that maintain life
take place much faster than they would outside a living body. This
is because living tissue manufactures substances called enzymes each
of which has the property of speeding up a certain chemical reaction
required by the organism. We know this because enzymes may be
removed from the tissues that made them, and can then be used to
accelerate the same reactions in test tubes or factory vats.

A number of enzymes have been obtained in the pure state. The
digestive enzymes, such as pepsin, trypsin, and diastase, are prob-
ably best known because their action on food is easily seen. Pepsin
added to milk will curdle it and if conditions are made to simulate
those in the stomach, the curd will later dissolve. Trypsin, from the
pancreas, acts in much the same way; a solution containing it dis-
solves a piece of cooked meat rather quickly. Diastase, found in
human saliva, changes starch to sugar. Each type of food is digested
hy a particular class of enzymes.

Enzymes like those in the animal digestive tract are also found
elsewhere. The poison of the rattlesnake contains a protein-digesting
enzyme. Certain plants also contain such enzymes. Small quan-
tities of protein-digesting enzymes have been found in the tissues of
higher animals and fish. Thus stored meat and fish undergo self-
digestion which if allowed to continue too long makes the flesh soft
and finally unpleasant to eat.

416139°— 41

Z CIRCULAR 631, U. S. DEPARTMENT OF AGRICULTURE

Enzymes find curious uses: In the brewing industry, diastase ob-
tained from malt is used to transform starch to sugar; starch may be
quickly removed from cloth by use of the same reaction.

The Bankhead-Jones funds for special research have enabled the
Department of Agriculture to investigate many of these enzymes.
Knowledge of their behavior has resulted in improved ways of han-
dling the perishable products of agriculture, and has solved many
technical difficulties for the miller, baker, brewer, canner, frozen-food
distributor, and even the laundryman.

This, circular describes a series of studies on protein-digesting
enzymes of the pineapple and papaya fruits, including a new method
of preparation that stabilizes their valuable properties. It discusses
their action in tenderizing meat and describes their preparation in
crystalline form.

SOURCES OF PAPAIN AND BROMELIN

In handling certain fruits, such as figs, cantaloups, pineapples,
and green papayas, workers' hands sometimes become sore, partic-
ularly around the fingernails. The injury to the skin is caused by
a protein-digesting enzyme in the juice or in the latex of the plant.

The presence of a protein-digesting substance in the leaves and
green fruit of the papaya tree (Carica Papaya) has long been known.
The existence of a similar meat-digesting enzyme in the juice of the
pineapple, was first noted by Marcano of Venezuela in comparatively
recent times. The protein-digesting (or proteolytic) principle has
now been prepared from both papaya and pineapple and may be
stored, shipped, and in general used without reference to the materia]
of its origin. Papain, the name of the material obtained from the
papaya fruit, has found employment in medicine, in industry, and
as a meat tenderizer.

It is recorded that many years ago the West Indian peoples pressed
pieces of meats between papaya leaves in order to make the meat
tender. In Barbados, lumps of the green fruit were added to an
otherwise intractable meat stew.

Bromelin, a substance similar to papain but concentrated from raw
pineapple juice is still a scientific curiosity and little used. Present
information about bromelin indicates that it would serve about the
same purposes as papain, although there are slight differences in
action that might make it more or less suitable for particular cases.

Many plants besides those mentioned contain demonstrable quan-
tities of protein-digesting enzymes, although few are known in which
the quantity of enzyme is so large. Proteinases in figs, milkweed,
the seeds of lima beans, and the kernels of wheat have been studied
and found to be related to papain and bromelin. They have been
given the general name of papainases. The proteolytic enzyme in
the pumpkin belongs to another group. Insect-eating plants such
as the pitcherplant and the sundew contain proteinases with still
other characteristics, whose purpose is apparently to digest the bodies
of captured insects.

In trade, papain and bromelin are names given to the material pre-
pared respectively from the papaya and pineapple fruits and are

PROTEIN-DIGESTING ENZYMES OF PAPAYA AND PINEAPPLE 6

merely concentrates containing the active principle. In chemical work
the names are used to refer to the active principles themselves. Pa-
pain, but not bromelin, has been obtained in the pure state. In each
case the active principle is a protein-digesting enzyme — that is,
a substance capable of digesting meat and other proteins in the same
way that they are digested by the pepsin and trypsin of the digestive
tract of man and animals.

PREPARATION OF PAPAIN

Commercial papain is simply the dried latex of the green papaya
fruit. In order to obtain the latex the skin of the fruit is scratched
with a sliver of glass or a sharp piece of bone. The latex runs out
and either drips down into a cup held under the fruit, or clots on
the skin to a pasty mass that is scraped into the cup. The latex is
then dried and powdered. Figure 1 shows the collection of latex from
green papaya.

Latex is always taken while the fruit hangs on the tree because
the scratching operation may be repeated as often as the plant is
able to replenish the supply of latex and as long as the fruit is green.
Green papaya fruit contains the largest quantity of latex. Kipe
fruit yields little latex and appears to be devoid of enzyme. Claims
sometimes made for the digestive action of juice or pulp from the
ripe papaya fruit should be greatly discounted, because the enzyme
disappears during ripening. Many products made from ripe papaya
fruit have been examined in this Laboratory, but none has ever
shown any protein-digesting activity. In the pineapple, however,
ripening does not destroy the enzyme, and ripe pineapple or the
juice from it contains plenty of active bromelin before it is heated.
This explains why raw pineapple juice cannot be used to make gelatine
desserts. The gelatine is digested by the enzyme. It may also ex-
plain the old-time use of fresh pineapple as a remedy for "clearing
out" a sore throat, as it seems quite probable that dead tissue and
mucous would be digested by the enzyme, whereas healthy live tissue
is not so easily affected.

Studies made at the Hawaiian Agricultural Experiment Station
showed that much of the enzyme in fresh papaya latex disappeared
during the drying process, no matter how carefully conducted. This
is partly due to the loss from the latex of another substance that
serves as an activator of the enzyme. The chemical constitution of
this activator is not known, but it contains an organic sulfide group
and belongs chemically to the class of substances known as reducing
agents. The activator appears to keep the enzyme in a reduced con-
dition, for if the enzyme is oxidized it becomes inactive but may be
reactivated by the addition of this natural activating substance or
by the addition of many chemicals that are good reducing agents.
Among the substances that may be used for this purpose are cysteine,
sulfites, alkaline cyanides, and sulfides, although it must be remem-
bered that the last two are extremely poisonous chemicals. In fact,
almost any good reducing agent that does not destroy proteins seems
able to serve as an activator of papain. It is probable therefore that
exposure to air inactivates papain because the enzyme is oxidized.

4

CIRCULAR 63 1, U

DEPARTMENT OF AGRICULTURE

In order to avoid the oxidation of the enzyme in air and the result-
ant loss of its activity, a method has been worked out in the Depart -

Figure 1. — Collecting latex from green papaya.

ment of Agriculture in which salt is added to the moist latex, which is
thereafter allowed to dry down to a thick paste. This salted paste

PROTEIN-DIGESTING ENZYMES OF PAPAYA AND PINEAPPLE O

was found to contain almost all the activity of the original latex
and when kept in closed containers for 7 months it did not deteriorate
appreciably. The same amount of latex prepared by the older meth-
ods would have had at best something less than half the enzymic
activity of the salted paste.1 The details are given in a technical
publication.2 Table 1, however, summarizes the results of an experi-
ment.

Table 1. — Percentage of the original activity 1 of the undried latex remaining
after the storage of vacuum-dried and partly dried preparations of latex at
room temperature.

Activated enzyme after
storage of—

Naturally active enzyme
after storage of —

Preparation

0
days

33-

36

days

45-

50

days

130-
135
days

190

days

0
days

33-
36

days

45-

50

days

130-
135
days

190
days

Dried latex. _ ._. .. ..

Per-
cent
92

85

78

1 82
I 100

Per-
cent

80

58

Per-
cent
41

49

59

80

Per-
cent

Per-
cent

Per-
cent
91

37

76

73
30

Per-
cent

74
19

Per-
cent

17

34
55

74

Per-
cent

Per-
cent

Dried latex containing 10 percent of
NajS

Dried latex containing 10 percent of
NaCl

Partly dried latex:

Papaya solids 35 percent. _ _ _

74

75

67

NaCl 20 percent

Water 45 percent

65

Partly dried latex:

NaaS 10 percent.

NaCl 10 percent. ...

i The activity of the original latex was 1.1-1.3 milk-clotting units per milligram of dry weight without
activation and 1.2-1.5 units after activation with sodium cyanide. The percent activity is calculated on
the weight of papaya solids only, thus excluding such additions as salt and sulfide.

CRYSTALLINE PAPAIN

The Department of Agriculture has crystallized two pure enzymes
from papaya latex. These two enzymes have been named papain and
chymopapain. The crystals are the enzyme proteins and are naturally
more active than any other known preparations. However, thej^
sometimes require the addition of one of the activating substances
mentioned previously, and may also contain some inert protein. It
is naturally important in making crystalline papain to have as little
inert protein present as possible and methods to minimize it have
been worked out. They depend on preventing the oxidation of the
papain during the preparation of the crystals. One method of pre-
paring the crystals is to extract the moist clotted latex with very
dilute sodium cyanide and to precipitate the enzyme from the ex-
tract with ammonium sulfate. Repeated precipitation of the en-
zyme in the cold by 0.4 saturation with ammonium sulfate eventually
results in a crystalline protein, which does not change in activity on
repeated recrystallization. The crystals are usually fine needles, but
large plates with six-sided faces have also been obtained. The two

1 United States patent dedicated to the use of the public has been applied for.

2 Balls, A. K., Lineweaver, H., and Schwimmer, S. drying of papaya latex, stabil-
ity op papain. Indus, and Engin. Chem. 32 : 1277. 1940.

6 CIRCULAR 63 1, U. S. DEPARTMENT OF AGRICULTURE

forms are shown in figure *2. For details of preparation the original
paper should be consulted/"

LV /2

>&i<--; -

i

r m

7

V"

w- :$& a'K-

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(A) {B)

Figure 2. — A, Crystalline papain, needle form. X 400 : B, crystalline papain, plate

form. X 100.

Another method of minimizing oxidation is to add an acid to the
papaya latex. This treatment destroys the papain, but it enables
the chemist to obtain the chymopapain, which is later precipitated
with common salt and then crystallized. It may be seen from the
method of preparation that chymopapain is resistant to rather strong
acid, something most enzymes are not, so interesting uses of this
enzyme are to be expected.

PROPERTIES AND USES OF PAPAIN

Papain is a typical protein-digesting enzyme and like other fer-
ments of this class clots milk. The milk-clotting action of papain
has been investigated and can now be used as a method of measuring
the quantity of enzyme in a solution. The clotting time of a given
quantity of milk is inversely proportional to the quantity of enzyme :
that is, the more enzyme added to the milk, the shorter the time re-
quired. The details of this method of assay are given in a technical
paper.4

Although commerical papain is still usually sold by the pound
regardless of its enzymic activity, it should be sold on the basis of
activity, as this is what the purchaser really wishes to buy. For this
reason easy methods of measuring the activity are quite important.

The action of papain in clotting milk is similar to that of the en-
zyme rennin. which is used in making cheese. The cheese produced
from the papain curd is somewhat softer, however. The papain prob-
ably softens the curd by partly digesting it. Other plant proteinases
may be used to make curd and the juice of the fig is sometimes used for
this pin pose, particularly in the Balkans. Figs contain an enzyme
called ficin that is very like- papain.

"Balls. A. K., and Lixeweaver, Hans, isolation and properties of crystalline
papain. Jour. Biol. ('hem. 130 : 669-686, illus. 1939.

4 Balls, A K.. and HOOVER, Sam R. thi: mii.k-ci.ottim, action <>f papain. Jour.
Biol. Chem. 121 : T.;7-745. illus. 1937.

PROTEIN-DIGESTING ENZYMES OF PAPAYA AND PINEAPPLE 7

Another useful property of papain is its ability to disintegrate pro-
teins in a slightly acid environment. Many proteins are insoluble in
slightly acid liquids. One of the changes observed during the di-
gestion is that the protein becomes soluble. On account of this
property papain has been used for many years in a process for
making "chillproof" beer. Some of the protein of the grain is apt
to occur in the beer in a form that while soluble at room temperature,
is precipitated on cooling. The chilled beer is then no longer per-
fectly clear. The addition of small quantities of papain to such beer
digests the precipitable protein fragments so that they no longer
separate out in the cold.

Bacteriologists of the Department of Agriculture have found upon
investigation that commercial papain preparations often contain large
numbers of both aerobic and anaerobic bacteria. While not neces-
sarily harmful, their presence points definitely to the need of better
sanitation in the manufacture of these products. There is no rea-
son why papain should be contaminated if proper sanitary precau-
tions are taken. Nor is there any reason to suppose that the salt-
paste preparation developed by the Department would be any freer
from bacteria than other preparations unless proper care were taken
in making it. There is less chance of contamination in the crystalline
enzymes, but the use of the crystalline papains obviously lies in the
medical field because the preparations are too expensive for other
purposes. It has long been said that papain kills intestinal worms,
and furthermore digests the dead parasites. Papain is also said to
be an energetic blood-clotting agent and the purified material has
been used to stop bleeding. Studies on the medicinal use of these
enzymes have been carried on outside the Department of Agriculture.
So far there is no general agreement as to the results, and until there
is it is better to withhold judgment in regard to the pharmacological
efficacy of this material.

TENDERIZING MEAT

Much of the papain now imported into the United States is used
in preparations for tenderizing meat. These preparations are solu-
tions or suspensions of the enzyme, usually in some bacteriostatic
medium, such as dilute alcohol. They are smeared on the meat before
it is cooked and sometimes let into the tissues by stabbing with a
fork. After a few minutes the meat is cooked in the usual way.

The Department of Agriculture has investigated the changes that
the meat undergoes when treated with the enzyme. Both connective
tissue and muscle fibers were observed to be slightly digested, thus
producing a definite softening of the flesh.

Tests have shown that the enzyme always attacks the meat from the
surface, working inward. That is, the enzyme does not penetrate a
piece of meat and then work on the inside (fig. 3). For this reason,
ground beef is more evenly affected than beefsteak. Some method of
distributing the enzyme throughout the meat (as by puncturing the
latter with a fork) seems to be necessary for good results. Papain is
relatively resistant to heat, and most of the action on meat occurs
during cooking. Thus, an overdose of enzyme at too long a cooking
period may even cause too great a disintegration of the meat. On the

8 CIRCULAR 631, U. S. DEPARTMENT OF AGRICULTURE

other hand, if the temperature is too high, the enzyme is destroyed,
and its action stops. The greatest break-down of the tissue occurs
when there is intimate contact between the enzyme and the meat
fibers at about 70° C which is a moderate cooking temperature.
The pure enzymes produce the same effect as the commercial prepara-
tions, showing that the effect is due to the enzyme action rather than
to the bacteria with which the commercial products are often con-
taminated.

One of the difficulties encountered by the manufacturers of meat
tenderizers is that such preparations lose their potency in a compara-
tively short time. This is probably due in great part to oxidation.
It is hoped that the salt-paste papain may obviate some of this
difficulty.

B

Figure 3. — Vertical cross sections of two pieces of beefsteak cooked together
in an oven for the same length of time. After cooking each piece was washed
in running water for a minute. (A) Was untreated: (B) received 1 drop
of a strong papain solution placed on the upper surface just before cooking.
Note the crater which indicates where the enzyme solution bad liquefied the
meat, the liquefied portion being later removed by the running water.

BROMELIN

Bromelin, obtained from the pineapple, is a proteolytic enzyme
similar to but not identical with papain. Like papain, its action
is aided by the presence of a reducing agent such as hydrogen sul-
fide, cysteine, sodium cyanide, or the natural activator that occurs with
the enzyme in the pineapple. As yet bromelin has been little used,
although it was reported many years ago that meat packers employed
this material to prepare "meat jelly." It has never been prepared
in anything approaching the pure state and is not at present available
in commercially significant quantities. Much less is known of the
properties of bromelin than of papain, but there is no reason to
suppose that its effects would be essentially different. Apparently
it is not as efficient in clotting milk as papain but seems to break
down proteins about as thoroughly. It has anthelmintic or worm-
killing properties similar to those of papain.

PROTEIN-DIGESTING ENZYMES OF PAPAYA AND PINEAPPLE 9

Since bromelin occurs in the ripe pineapple as well as in the green
plant it may be obtained from ripe pineapple juice that has not been
heated. The Department of Agriculture, in cooperation with the
Hawaiian Agricultural Experiment Station, has developed a method
for obtaining this enzyme from the peelings and cores of the fruit.
In the process of canning pineapples such material is pressed to re-
move the juice. The fibrous solid material is then dried and sold
for cattle feed. Mild pressure is first applied and yields a high-grade
juice that is used as a food product. Greater pressure then yields a
medium-quality juice, and still greater pressure yields a juice of
poor quality. This third press-juice is not particularly valuable to
the pineapple canner but it contains considerable sugar, citric acid,
and bromelin. It would be uneconomical to sacrifice the sugar in
order to obtain the enzyme, but the latter may be precipitated by
the addition of alcohol, leaving the sugar in solution. The alcohol
may then be distilled off and the sugar fermented with yeast to more
alcohol, which in turn is removed by another distillation. In this
way both the enzyme and the alcohol equivalent of the sugar may be
obtained from the poor grade of press juice.5

Bromelin also exists in the juice of pineapple leaves and stalks,
but it is hard to press out since the tissues are very tough and fibrous
and contain little liquid. It is therefore doubtful whether any satis-
factory method can be devised to use the leaves and stalks as a
source of the enzyme.

Experiments have shown that bromelin is about as effective as
papain in tenderizing meat. Like papain, the pineapple enzyme
attacks both muscle fibers and connective tissues. In these experi-
ments preparations of the enzyme were applied in the same manner
as was previously described for papain. The odor of bromelin prep-
arations offers a favorable contrast to those of papain.

SUMMARY

The preparation and present uses of papain as well as the prospec-
tive uses of bromelin are discussed. A method of obtaining bromelin
from pineapple juice without losing the value of the sugar has been
suggested. The use of papain in tenderizing meat and the possible
use of bromelin for a similar purpose are discussed.

A method developed by the Department of Agriculture is de-
scribed for the manufacture of a cleaner and more active preparation
of commercial papain than that ordinarily sold today. This method
has at least doubled the yield of enzyme from a papaya tree because
it prevents the loss of enzyme that takes place in the present process
of manufacture.

Methods have also been found to prepare two proteolytic enzymes
in the crystalline form from crude papain. These preparations are
too expensive for ordinary use, but they may be of service in medicine.

5 Balls, A. K., Thompson, R. R., and Kies, M. W. bromelin : properties and com-
mercial production. Indus, and Engin. Chem. 33 : 950-953, illus. 1941.

ORGANIZATION OF THE UNITED STATES DEPARTMENT OF AGRICULTURE
WHEN THIS PUBLICATION WAS EITHER FIRST PRINTED OR LAST REVISED

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periment Stations.

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